HiPerFect Transfection Reagent Handbook

Transcription

1 Fifth Edition October 2010 HiPerFect Transfection Reagent Handbook For transfection of eukaryotic cells with sirna and mirna Sample & Assay Technologies

2 QIAGEN Sample and Assay Technologies QIAGEN is the leading provider of innovative sample and assay technologies, enabling the isolation and detection of contents of any biological sample. Our advanced, highquality products and services ensure success from sample to result. QIAGEN sets standards in: Purification of DNA, RNA, and proteins Nucleic acid and protein assays microrna research and RNAi Automation of sample and assay technologies Our mission is to enable you to achieve outstanding success and breakthroughs. For more information, visit

3 Contents Kit Contents 5 Shipping and Storage 5 Quality Control 5 Product Use Limitations 5 Product Warranty and Satisfaction Guarantee 6 Technical Assistance 6 Safety Information 7 Introduction 8 Principle and procedure 8 Protocols for various cell types and for special applications 10 Reagents to Be Supplied by User 13 Important Notes 14 Calculating concentrations of sirna 14 Optimizing sirna transfection 14 Calculating sirna and reagent for different formats 16 Transfection in multiwell plates preparing a master mix 17 Performing appropriate RNAi control experiments 17 Monitoring gene silencing at the mrna or protein level 18 Protocols for adherent cells Fast-Forward Transfection of Adherent Cells with sirna/mirna in 24-Well Plates 20 Reverse Transfection of Adherent Cells with sirna/mirna in 96-Well Plates 22 Reverse Transfection of Adherent Cells with sirna/mirna in 384-Well Plates 24 Transfection of Adherent Cells with sirna/mirna (Traditional Protocol) 26 Protocols for suspension cells and macrophages Transfection of Suspension Cell Lines, Including Jurkat and K562, with sirna/mirna in 24-Well Plates 28 Transfection of Macrophage Cell Lines, Including J774.A1 and RAW 264.7, with sirna/mirna in 24-Well Plates 30 Transfection of Differentiated Macrophage Cell Lines, Including THP-1, with sirna/mirna in 24-Well Plates 32 HiPerFect Transfection Reagent Handbook 10/2010 3

4 Protocols for primary cells Transfection of HUVEC Cells with sirna/mirna in 24-Well Plates 34 Transfection of Fibroblasts with sirna/mirna in 24-Well Plates 36 Transfection of Keratinocytes with sirna/mirna in 24-Well Plates 38 Transfection of Epithelial Cells with sirna/mirna in 24-Well Plates 40 Transfection of Smooth Muscle Cells with sirna/mirna in 24-Well Plates 42 Guidelines for Transfection of Primary Neurons with sirna/mirna in 96-Well Plates 44 Guidelines for Transfection of Primary Hepatocytes with sirna/mirna in 96-Well Plates 46 Protocols for special applications Large-Scale Transfection of Adherent Cells with sirna/mirna in 100 mm dishes 48 Long-Term Transfection of Adherent Cells with sirna/mirna 50 Troubleshooting Guide 52 Appendix A: Optimizing sirna Transfection of Suspension and Macrophage Cells 55 Appendix B: Recommendations for Primary Cells 59 References 62 Ordering Information 63 4 HiPerFect Transfection Reagent Handbook 10/2010

5 Kit Contents HiPerFect Transfection 0.5 ml 1 ml 4 x 1 ml 100 ml Reagent Catalog no Transfections per kit Up to 166 Up to 333 Up to Up to (in 24-well plates) well plates HiPerFect Transfection 0.5 ml 1 ml 4 x 1 ml 100 ml Reagent Handbook Shipping and Storage HiPerFect Transfection Reagent is supplied as a ready-to-use solution and is shipped at ambient temperature without loss of stability. However, it should be stored at 2 8 C upon arrival. HiPerFect Transfection Reagent does not need to be stored on ice during the transfection procedure. Quality Control In accordance with QIAGEN s ISO-certified Quality Management System, each lot of HiPerFect Transfection Reagent is tested against predetermined specifications to ensure consistent product quality. Product Use Limitations The HiPerFect Transfection Reagent is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease. All due care and attention should be exercised in the handling of the products. We recommend all users of QIAGEN products to adhere to the NIH guidelines that have been developed for recombinant DNA experiments, or to other applicable guidelines. HiPerFect Transfection Reagent Handbook 10/2010 5

6 Product Warranty and Satisfaction Guarantee QIAGEN guarantees the performance of all products in the manner described in our product literature. The purchaser must determine the suitability of the product for its particular use. Should any product fail to perform satisfactorily due to any reason other than misuse, QIAGEN will replace it free of charge or refund the purchase price. We reserve the right to change, alter, or modify any product to enhance its performance and design. If a QIAGEN product does not meet your expectations, simply call your local Technical Service Department or distributor. We will credit your account or exchange the product as you wish. Separate conditions apply to QIAGEN scientific instruments, service products, and to products shipped on dry ice. Please inquire for more information. A copy of QIAGEN terms and conditions can be obtained on request, and is also provided on the back of our invoices. If you have questions about product specifications or performance, please call QIAGEN Technical Services or your local distributor (see back cover or visit ). Technical Assistance At QIAGEN we pride ourselves on the quality and availability of our technical support. Our Technical Service Departments are staffed by experienced scientists with extensive practical and theoretical expertise in sample and assay technologies and the use of QIAGEN products. If you have any questions or experience any difficulties regarding HiPerFect Transfection Reagent or QIAGEN products in general, please do not hesitate to contact us. QIAGEN customers are a major source of information regarding advanced or specialized uses of our products. This information is helpful to other scientists as well as to the researchers at QIAGEN. We therefore encourage you to contact us if you have any suggestions about product performance or new applications and techniques. For technical assistance and more information, please see our Technical Support center at or call one of the QIAGEN Technical Service Departments or local distributors (see back cover or visit ). 6 HiPerFect Transfection Reagent Handbook 10/2010

7 Safety Information When working with chemicals, always wear a suitable lab coat, disposable gloves, and protective goggles. For more information, please consult the appropriate material safety data sheets (MSDSs). These are available online in convenient and compact PDF format at where you can find, view, and print the MSDS for each QIAGEN kit and kit component. 24-hour emergency information Emergency medical information in English, French, and German can be obtained 24 hours a day from: Poison Information Center Mainz, Germany Tel: HiPerFect Transfection Reagent Handbook 10/2010 7

8 Introduction The application of RNA interference (RNAi) to mammalian cells has revolutionized the field of functional genomics. The ability to simply, effectively, and specifically downregulate the expression of genes in mammalian cells holds enormous scientific, commercial, and therapeutic potential. Efficient transfection of sirna is critical for effective gene silencing. HiPerFect Transfection Reagent has been developed for efficient transfection of cells even when low sirna concentrations are used. Highly efficient transfection and silencing have been observed in some cases using as little as 100 pm sirna. HiPerFect Transfection Reagent provides highly efficient sirna transfection over a range of sirna concentrations from low to high, allowing researchers to choose the sirna concentration they wish to use. HiPerFect Reagent is also highly suited to mirna mimic/mirna inhibitor transfection for mirna research. Principle and procedure Off-target effects in RNAi experiments Studies have indicated that transfection of sirna can result in off-target effects, in which sirnas affect the expression of nonhomologous or partially homologous gene targets. Off-target effects can include mrna degradation, inhibition of translation, or induction of an interferon response (1 4). The mechanisms of off-target effects are not fully understood. They may be caused by sirna targeting mrna with close homology to the target mrna, by sirnas functioning like mirnas, or by a cellular response to sirna toxicity. In addition, some researchers have observed an sirna-mediated interferon response. Research suggests that off-target effects, which may produce misleading results in RNAi experiments, can be largely avoided by using low sirna concentrations (5, 6). By allowing highly efficient sirna uptake and release within cells, HiPerFect Transfection Reagent enables gene silencing using low sirna concentrations without compromising knockdown efficiency. High knockdown and minimal risk of off-target effects in RNAi HiPerFect Transfection Reagent enables highly efficient sirna transfection, allowing gene silencing using low sirna concentrations without compromising knockdown efficiency. In the data shown (Figure 1), efficient knockdown (>80%) is achieved with sirna concentrations in the range of 1 50 nm. Transfection of 1 nm sirna resulted in 86% knockdown and transfection of 5 nm sirna increased the knockdown efficiency to 96%. Depending on the purpose of the RNAi experiment, the optimal concentration of sirna to use may be 1 nm (minimal risk of off-target effects and efficient knockdown) or 5 nm (higher knockdown efficiency). To achieve the highest possible knockdown levels, it may be necessary to increase sirna concentrations above the levels indicated in the optimization schemes in Tables 1 (page 15), 5 (page 55), 6 (page 56), and 11 (page 60). The optimal sirna concentration will also depend on sirna potency, cell type, and the target gene. 8 HiPerFect Transfection Reagent Handbook 10/2010

9 Relative expression of CDC2 mrna (%) HiPerFect Transfection Reagent Reagent L 0 Untransfected control 50 nm sirna 25 nm sirna 12.5 nm sirna 5 nm sirna 1 nm sirna Decreasing sirna concentration 25 nm Nonsilencing sirna Increasing silencing effect Figure 1. HiPerFect Reagent provides effective CDC2 knockdown even at low sirna concentrations. HeLa S3 cells were transfected with a range of concentrations of sirna targeted against CDC2 using HiPerFect Transfection Reagent from QIAGEN or Reagent L from another supplier. Nonsilencing sirna was also transfected. After 48 hours, cells were harvested and total cellular RNA was purified using the RNeasy system, and reverse transcribed using Omniscript Reverse Transcriptase. The resultant cdna was used for quantitative, real-time RT-PCR. Expression of CDC2 was normalized to expression of GAPDH. Values derived from real-time RT-PCR of control, untransfected cells were set at 100% and the relative expression levels of cells transfected with the experimental sirna are shown. mirna research micrornas (mirnas) are a class of endogenous small RNA molecules with similar characteristics to sirnas. In recent years, it has been discovered that mirnas play a role in many diverse biological processes such as development, differentiation, and apoptosis. Misregulation of mirna expression is reported to be associated with several cancers and other diseases. The mirna system is an endogenous mechanism of regulation of gene expression. Mature mirnas contribute to the regulation of endogenous genes, primarily by translational repression. In addition, mirnas can mediate mrna destruction by rapid deadenylation and/or decapping. Naturally occurring mirna-binding sites are typically found in the 3' untranslated regions (UTRs) of target mrnas. Their partial complementarity has made positive identification of true binding sites difficult and imprecise. HiPerFect Transfection Reagent Handbook 10/2010 9

10 Transfection of synthetic mirna mimics or mirna inhibitors are techniques used to elucidate the targets and roles of particular mirnas. mirna mimics are chemically synthesized mirnas which mimic naturally occurring mirnas after transfection into the cell. mirna inhibitors are single-stranded, modified RNAs which specifically inhibit mirnas. Reduced gene expression after transfection of an mirna mimic or increased expression after transfection of an mirna inhibitor provides evidence that the mirna is involved in regulation of that gene. Alternatively, the role of mirnas in various pathways can be studied by examination of a specific phenotype after mirna mimic or inhibitor transfection. Screening experiments can be performed to examine a phenotype after transfection of large numbers of synthetic mirna mimics or inhibitors. HiPerFect Transfection Reagent is ideally suited to both low- and high-throughput transfection of mirna mimics or mirna inhibitors. In addition to transfection, QIAGEN provides synthetic mirna mimics and mirna inhibitors, as well as innovative mirneasy and miscript technologies for mirna purification and detection. For more details, background information, and application data, visit Protocols for various cell types and for special applications This handbook contains protocols for adherent cells, suspension cells and macrophages, and primary cells. Protocols for adherent cells For adherent cells, two protocols are provided for sirna/mirna transfection in 24-well plates. In the Fast-Forward Protocol (page 20), cell seeding and transfection are carried out on the same day. In the Traditional Protocol (page 26), cell seeding is performed the day before transfection. The Fast-Forward Protocol is quicker and saves labor compared to the Traditional Protocol (Figures 2 and 3). For high-throughput experiments with adherent cells, protocols are provided for reverse transfection in 96-well plates and 384-well plates (page 22 and page 24, respectively). In these protocols, cells are seeded and transfected on the same day. sirna/mirna is spotted into wells followed by the addition of HiPerFect Reagent. After complex formation, cells are added to the wells. Reverse transfection is rapid and convenient, and is frequently used for high-throughput experiments (Figures 2 and 3). 10 HiPerFect Transfection Reagent Handbook 10/2010

11 Traditional Protocol Fast-Forward Protocol Reverse-Transfection Protocol Cell seeding Cell seeding Day 1 Complex formation Transfection Day 2 Complex formation Transfection Figure 2. The Fast-Forward and Reverse-Transfection Protocols save time and labor. In the Reverse-Transfection and Fast-Forward Protocols, cell seeding, complex formation, and transfection are all performed on the same day. In the Traditional Protocol, cell seeding is performed the day before transfection. Fast-Forward Protocol Reverse-Transfection Protocol Step 3. Cells Step 2. Transfection complexes Step 1. Cells Step 2. HiPerFect Reagent Step 1. Nucleic acid Figure 3. The order of steps differs between the Fast-Forward and Reverse Transfection Protocols. In the Fast- Forward Protocol, cells are added to plate wells first, followed by transfection complexes. In the Reverse- Transfection Protocol, sirna/mirna is added to plate wells, followed by HiPerFect Reagent. After complex formation in the wells, cells are added. HiPerFect Transfection Reagent Handbook 10/

12 Protocols for suspension cells and macrophages Protocols are also provided for sirna/mirna transfection of suspension cells (page 28), macrophages (page 30), and differentiated macrophages (page 32) in 24-well plates. Protocols for primary cells Protocols are provided for sirna/mirna transfection of HUVEC cells, fibroblasts, keratinocytes, epithelial cells, and smooth muscle cells in 24-well plates. In addition, guideline protocols are provided for transfection of primary neurons and primary hepatocytes in 96-well plates. These guidelines are standard parameters based on experiments with similar cell types and further optimization is recommended to achieve maximum transfection efficiency. The primary-cell protocols provided have been mainly tested with human cells. However, they are applicable to all cell types. Protocols for special applications with adherent cells For large-scale transfection, a protocol is provided for transfection in 100 mm dishes on page 48. In this protocol, cell seeding and transfection are performed on the same day. A protocol for long-term gene silencing is included on page 50. This protocol is for the study of phenotypic effects that require knockdown for periods longer than 3 4 days before they can be observed. Gene knockdown using sirna is transient and expression usually increases again several days after transfection, especially when the cells are subcultured. However, the long-term protocol allows gene knockdown for over two weeks. The protocol uses HiPerFect Transfection Reagent to deliver sirna/mirna every time the cells are diluted and replated. This is possible because transfection with HiPerFect Reagent ensures extremely low levels of cytotoxicity, so cells remain viable and healthy after multiple cycles of dilution and transfection. 12 HiPerFect Transfection Reagent Handbook 10/2010

13 Reagents to Be Supplied by User When working with chemicals, always wear a suitable lab coat, disposable gloves, and protective goggles. For more information, consult the appropriate material safety data sheets (MSDSs), available from the product supplier. Culture medium sirna of interest. We recommend functionally validated HP Validated sirna or HP GenomeWide sirna, which is available for every human, mouse, and rat gene. The GeneGlobe Web portal allows convenient ordering ( ). QIAGEN also provides custom synthesis of highly pure HPP Grade sirna and sirna Sets for high-throughput RNAi. For more information, visit mirna mimic or mirna inhibitor of interest. For information about mirna mimics and mirna inhibitors from QIAGEN, visit For transfection of differentiated macrophages (page 32): a differentiationinducing agent such as PMA For long-term transfection (page 50): Sterile PBS (1x PBS: 137 mm NaCl, 2.7 mm KCl, 4.3 mm Na 2 HPO 4, 1.47 mm KH 2 PO 4 ; adjust to a final ph of 7.4). Store at room temperature (15 25 C) Trypsin/EDTA (Hanks Balanced Salt Solution containing 0.05% (w/v) porcine trypsin and 0.5 mm EDTA, or another appropriate trypsin or enzymatic solution) HiPerFect Transfection Reagent Handbook 10/

14 Important Notes Calculating concentrations of sirna Approximate values for a double-stranded, 21 nt sirna molecule: 20 µm sirna is equivalent to approximately 0.25 µg/µl The molecular weight of a 21 nt sirna is approximately µg/nmol To calculate the amount of sirna to use for different formats, refer to Tables 3 and 4 for adherent cells (page 16), Table 8 for suspension cells (page 57), Table 9 for macrophages (page 57), Table 10 for differentiated macrophages (page 58), or Table 13 for primary cells (page 61). Optimizing sirna transfection To achieve the best results in sirna transfection of adherent cells, we recommend optimizing the following parameters. For details on optimizing sirna transfection in suspension or macrophage cells, see Appendix A (page 55). For details on optimizing sirna transfection in primary cells, see Appendix B (page 59). For appropriate amounts of mirna mimic/mirna inhibitor to transfect, refer to the manufacturer's instructions. Amount of sirna The amount of sirna used is critical for efficient transfection and gene silencing. The recommended starting concentration for transfection of sirna in 24-well plates is 5 nm. A pipetting scheme for optimizing sirna transfection of adherent cells in 24-well plates is shown in Table 1 (page 15). Ratio of HiPerFect Transfection Reagent to sirna The ratio of HiPerFect Transfection Reagent to sirna should be optimized for every new cell type and sirna combination used. As a starting point for optimization when using 24-well plates, we recommend 5 nm sirna and 3 µl HiPerFect Transfection Reagent for adherent cells. To optimize sirna transfection in 24-well plates, prepare separate transfection mixtures according to Table 1 (page 15). Please note that Table 1 is intended only as a guideline for starting amounts of sirna and reagent. These amounts worked well as a starting point for transfection optimization in the range of cell lines that have been tested using HiPerFect Transfection Reagent. If necessary, it is also possible to further reduce the volume of HiPerFect Transfection Reagent without significantly compromising performance. 14 HiPerFect Transfection Reagent Handbook 10/2010

15 Table 1. Pipetting scheme for optimizing transfection of adherent cells in 24-well plates* Amount (conc.) of sirna 75 ng 75 ng 75ng (10 nm) (10 nm) (10 nm) Volume of HiPerFect Reagent 1.5 µl 3 µl 4.5 µl Amount (conc.) of sirna 37.5 ng 37.5 ng 37.5 ng (5 nm) (5 nm) (5 nm) Volume of HiPerFect Reagent 1.5 µl 3 µl 4.5 µl Amount (conc.) of sirna 7.5 ng 7.5 ng 7.5 ng (1 nm) (1 nm) (1 nm) Volume of HiPerFect Reagent 1.5 µl 3 µl 4.5 µl * Amounts given are per well of a 24-well plate. sirna concentrations shown in brackets refer to the final sirna concentration in each well. For details on optimizing transfection in suspension or macrophage cells, see Appendix A (page 55) and in primary cells, see Appendix B (page 59). Cell density at transfection The optimal cell confluency for transfection should be determined for every new cell type to be transfected and kept constant in future experiments. This is achieved by counting cells before seeding and, in the case of the Traditional Protocol (page 26), by keeping the interval between seeding and transfection constant. This ensures that the cell density is not too high and that the cells are in optimal physiological condition at transfection. The recommended number of cells to seed for different formats is shown in Table 2. Table 2. Recommended number of adherent cells to seed for different formats Culture format Fast-Forward Protocol Reverse-Transfection Protocol Traditional Protocol (day of transfection) (day before transfection) 384-well plate , well plate 1 5 x x well plate 2 8 x x well plate x x well plate x x well plate x x mm dish x x mm dish 2 4 x x 10 6 For recommended numbers of suspension or macrophage cells to seed, see Appendix A (page 55) and for primary cells, see Appendix B (page 59). HiPerFect Transfection Reagent Handbook 10/

16 Calculating sirna and reagent for different formats Table 3 and Table 4 give starting points for optimization of sirna transfection of adherent cells in different plate and dish formats for the Fast-Forward Protocol (page 20) and the Traditional Protocol (page 26). Starting points for 96-well, 384-well, and 100 mm formats are given in the respective protocols (pages 22 25, page 48). Table 3. Starting points for optimizing transfection of adherent cells in different formats Volume of Final Volume of medium sirna volume of HiPerFect Final Culture on cells amount diluted Reagent sirna format (µl) (ng)* sirna (µl) (µl) conc. (nm) 48-well plate well plate well plate well plate mm dish * Table 4 shows the corresponding volumes of sirna stock to use for each sirna amount. For starting points for optimizing transfection of suspension or macrophage cells, see Appendix A (page 55) and for primary cells, see Appendix B (page 59). Table 4. Volumes of sirna stock for different sirna amounts Equivalent volume of Equivalent Equivalent sirna 0.2 µm volume of volume of Culture amount sirna stock 2 µm sirna 20 µm sirna format (ng) (µl)* stock (µl)* stock (µl)* 48-well plate well plate well plate well plate mm dish * To make a 0.2 µm sirna stock, dilute 10 µl of a 20 µm stock to a final volume of 1000 µl. To make a 2 µm sirna stock, dilute 10 µl of a 20 µm stock to a final volume of 100 µl. 16 HiPerFect Transfection Reagent Handbook 10/2010

17 Transfection in multiwell plates preparing a master mix If you are performing transfection in multiwell plates, prepare a master mix of transfection complexes or of transfection reagent and culture medium (depending on the protocol) for distribution into plate wells. Calculate the required volumes of each component and the total volume before you prepare the master mix. Prepare 10% more master mix than is required to allow for pipetting errors (i.e., for a 48-well plate, prepare enough master mix for 53 wells). Add and mix the components of the master mix according to the instructions in the protocol. Use a repeat pipet to distribute the master mix. Performing appropriate RNAi control experiments It is important to perform suitable control experiments so that results can be correctly interpreted. A full range of control sirnas is available at For information about appropriate mirna control experiments, visit We recommend the following control experiments. Positive control sirna This is an sirna that is known to provide high knockdown of its target gene. For example, Mm/Hs_MAPK1 Control sirna (cat. no ) efficiently knocks down human and mouse MAPK1 and Rn_Mapk1 Control sirna (cat. no ) efficiently knocks down rat MAPK1. AllStars Hs Cell Death Control sirna (cat. no ) provides high knockdown of ubiquitous human cell survival genes which results in a high degree of cell death which is visible by light microscopy. These sirnas can be used as positive controls. A positive control is used to establish that the experimental set up for transfection and knockdown analysis is working optimally. An sirna that knocks down a gene resulting in the phenotypic effect under study may also be used as a positive control to ensure that the phenotypic assay is working optimally. A positive control sirna should be transfected in every RNAi experiment. Negative control sirna A negative control sirna should be a nonsilencing sirna with no homology to any known mammalian gene, such as AllStars Negative Control sirna (5 nmol) from QIAGEN (cat. no ). AllStars Negative Control sirna is the most thoroughly tested and validated negative control available. It has been shown to provide minimal nonspecific effects on gene expression and phenotype and is incorporated into RISC. For more details and to view data, visit HiPerFect Transfection Reagent Handbook 10/

18 Transfection of negative control sirna is used to determine whether changes in phenotype or gene expression are nonspecific. A negative control sirna should be transfected in every RNAi experiment. Transfection control sirna This control is used to measure the transfection efficiency. Transfection efficiency can be measured in several ways, for example by fluorescence microscopy after transfection of a fluorescently labeled sirna or by observation of the level of cell death after transfection of sirna that targets essential cell survival genes, such as AllStars Hs Cell Death Control sirna. sirna transfection efficiency should be as high as possible. This control should be performed for optimization, for example, when establishing RNAi in a new cell line. Mock transfection control Mock-transfected cells go through the transfection process without addition of sirna (i.e., cells are treated with transfection reagent only). This control is used to determine any nonspecific effects that may be caused by the transfection reagent or process. Untransfected cells control Gene expression analysis should be carried out on cells that have not been treated to allow measurement of the normal, basal level of gene expression. Results from untreated cells can be used for comparison with results from all other samples. Untreated cells should be analyzed in every RNAi experiment. Additional sirnas for phenotype confirmation A phenotypic effect caused by knockdown of a gene must be confirmed using at least one additional sirna targeted against a different area of the mrna. Monitoring gene silencing at the mrna or protein level Gene silencing can be monitored at either the mrna or the protein level. Protein analysis can be performed using western blotting, immunofluorescence, or FACS analysis. More information about protein analysis and a protocol for western blotting can be found at The AllPrep RNA/Protein Kit (cat. no ) allows simultaneous purification of RNA and protein from the same sample for streamlined downstream analysis after knockdown. Protein analysis is the most comprehensive way of showing that a gene has been downregulated. However, it may not always be possible to perform protein analysis (e.g., if antibodies for the protein of interest are not available for western blotting). 18 HiPerFect Transfection Reagent Handbook 10/2010

19 Silencing is usually monitored at the mrna level by real-time RT-PCR, microarray analysis, or northern blotting. Information about working with RNA and a northern blotting protocol are available at Quantitative, real-time RT-PCR is an easy and routinely used method to monitor gene silencing at the mrna level. QuantiTect Primer Assays are bioinformatically validated primer sets that are available for every human, mouse, rat, dog, drosophila, chicken, and arabidopsis gene. QuantiTect Primer Assays are used in combination with QuantiTect or QuantiFast SYBR Green Kits for sensitive and specific one-step or two-step real-time RT-PCR using SYBR Green detection. Expression data should be compared with levels of a housekeeping gene, such as GAPDH, to normalize for variable amounts of RNA in different samples. HiPerFect Transfection Reagent Handbook 10/

20 Fast-Forward Protocol Protocol: Fast-Forward Transfection of Adherent Cells with sirna/mirna in 24-Well Plates This protocol is provided as a starting point for optimization of sirna/mirna transfection of adherent cells in a single well of a 24-well plate using HiPerFect Transfection Reagent. This protocol can also be used as a starting point for optimizing transfection in other formats (e.g., 48-well plate, 12-well plate, 6-well plate, and 60 mm dish formats) using the sirna and reagent amounts listed in Table 3 on page 16. In this protocol, cell plating and transfection are performed on the same day. For a few sensitive cell types, it may be necessary to use the Traditional Protocol, where cells are plated the day before transfection (page 26). Note: We recommend when using 24-well plates that transfection is performed in the order described in this Fast-Forward Protocol, with cells seeded in wells first followed by addition of sirna/mirna reagent complexes. This ensures optimal mixing of cells and complexes. However, transfection can be performed using a Reverse-Transfection Protocol, with complexes added to wells and cells added on top of complexes, if desired. To perform a Reverse-Transfection Protocol, simply change the order in which cells and complexes are added to the plate (steps 1 5). Important points before starting Cells should be in optimal physiological condition at the time of transfection. The optimal amount of cells seeded depends on the cell type and time of analysis. If sirna has been ordered from QIAGEN, it is delivered lyophilized and must be resuspended prior to transfection.to resuspend, follow the instructions provided with the sirna. Nucleic acid amounts in the protocol refer to sirna. For appropriate amounts of mirna mimic/mirna inhibitor to transfect, refer to the manufacturer's instructions. Where possible, dilute mirna mimic/mirna inhibitor in the same volume as recommended for sirna in the protocol. Procedure 1. Shortly before transfection, seed x 10 5 cells per well of a 24-well plate in 0.5 ml of an appropriate culture medium containing serum and antibiotics. Cells may alternatively be seeded after step 3 of this protocol. 2. For the short time until transfection, incubate the cells under normal growth conditions (typically 37 C and 5% CO 2 ). 3. Dilute 37.5 ng sirna in 100 µl culture medium without serum (this will give a final sirna concentration of 5 nm after adding complexes to cells in step 5). Add 3 µl of HiPerFect Transfection Reagent to the diluted sirna and mix by vortexing. 20 HiPerFect Transfection Reagent Handbook 10/2010

21 IMPORTANT: The amount of HiPerFect Transfection Reagent and sirna required for optimal performance may vary, depending on the cell line and gene target. For details on optimization of the sirna to HiPerFect Transfection Reagent ratio, see page Incubate the samples for 5 10 min at room temperature (15 25 C) to allow the formation of transfection complexes. 5. Add the complexes drop-wise onto the cells. Gently swirl the plate to ensure uniform distribution of the transfection complexes. 6. Incubate the cells with the transfection complexes under their normal growth conditions and monitor gene silencing after an appropriate time (e.g., 6 72 h after transfection, depending on experimental setup). Change the medium as required. Note: The optimal incubation time for gene silencing analysis depends on the cell type, the gene targeted, and the method of analysis. This can be determined by performing a time-course experiment. When using fluorescently labeled sirna, microscopic analysis should be performed 4 24 h after transfection. Fast-Forward Protocol HiPerFect Transfection Reagent Handbook 10/

22 96-Well Plates Protocol: Reverse Transfection of Adherent Cells with sirna/mirna in 96-Well Plates This protocol is provided as a starting point for optimization of sirna/mirna transfection of adherent cells in a single well of a 96-well plate using HiPerFect Transfection Reagent. Starting points for optimizing transfection in other formats are listed in Table 3 on page 16. In this protocol, cell plating and transfection are performed on the same day. Note: Transfection in 96-well plates can also be performed using a Fast-Forward Protocol, if desired. In this Reverse-Transfection Protocol, complex formation takes place in the plate wells first and then cells are added on top of the complexes. In the Fast- Forward Protocol, cells are added to plate wells followed by complexes (see Figure 3, page 11). To perform a Fast-Forward Protocol, simply change the order in which cells and complexes are added to the plate (steps 1 4). However, we recommend using this Reverse-Transfection Protocol when working with 96-well plates because it is quicker, has fewer pipetting steps, and uses fewer materials (the Fast-Forward Protocol requires two plates, one for complex formation and one for adding complexes on top of cells; the Reverse-Transfection Protocol requires only one plate). Important points before starting Cells should be in optimal physiological condition at the time of transfection. The optimal amount of cells seeded depends on the cell type and time of analysis. If sirna has been ordered from QIAGEN, it is delivered lyophilized and must be resuspended prior to transfection.to resuspend, follow the instructions provided with the sirna. Nucleic acid amounts in the protocol refer to sirna. For appropriate amounts of mirna mimic/mirna inhibitor to transfect, refer to the manufacturer's instructions. Where possible, dilute mirna mimic/mirna inhibitor in the same volume as recommended for sirna in the protocol. Procedure 1. Spot 12.5 ng sirna in 1 3 µl of sirna Suspension Buffer/RNase-free water into a single well of a 96-well plate (this will give a final sirna concentration of 5 nm after addition of cells to complexes in step 4). Note: After this step, sirna may be stored at 20 C for long time periods. sirna may be stored in solution or may be dried in the plate at room temperature (15 25 C) before storage. Note: If preferred, sirna can be spotted in 25 µl of sirna Suspension Buffer/RNase-free water into each well. In this case, 150 µl culture medium (containing 1 5 x 10 4 cells) should be added in step HiPerFect Transfection Reagent Handbook 10/2010

23 2. Add 0.75 µl of HiPerFect Transfection Reagent to µl of culture medium without serum. Add the diluted HiPerFect Transfection Reagent to the prespotted sirna. Note: To ensure accurate pipetting, diluted HiPerFect Reagent should be prepared in a larger volume for use in multiple wells (see Transfection in multiwell plates preparing a master mix on page 17). Then add 25 µl of the dilution to a single well. IMPORTANT: The amount of HiPerFect Transfection Reagent and sirna required for optimal performance may vary, depending on the cell line and gene target. For details on optimization of the sirna to HiPerFect Transfection Reagent ratio, see page Incubate for 5 10 min at room temperature (15 25 C) to allow formation of transfection complexes. 4. Seed 1 5 x 10 4 cells in 175 µl of an appropriate culture medium (containing serum and antibiotics) into the well, on top of the sirna HiPerFect Reagent transfection complexes. 5. Incubate the cells with the transfection complexes under their normal growth conditions and monitor gene silencing after an appropriate time (e.g., 6 72 h after transfection, depending on experimental setup). Change the medium as required. Note: The optimal incubation time for gene silencing analysis depends on cell type, the gene targeted, and the method of analysis. This can be determined by performing a time-course experiment. 96-Well Plates HiPerFect Transfection Reagent Handbook 10/

24 Protocol: Reverse Transfection of Adherent Cells with sirna/mirna in 384-Well Plates This protocol is provided as a starting point for optimization of sirna/mirna transfection of adherent cells in a single well of a 384-well plate using HiPerFect Transfection Reagent. Starting points for optimizing transfection in other formats are listed in Table 3 on page 16. In this protocol, cell plating and transfection are performed on the same day. 384-Well Plates Important points before starting Cells should be in optimal physiological condition at the time of transfection. The amount of cells seeded depends on the cell type and time of analysis. If sirna has been ordered from QIAGEN, it is delivered lyophilized and must be resuspended prior to transfection.to resuspend, follow the instructions provided with the sirna. Nucleic acid amounts in the protocol refer to sirna. For appropriate amounts of mirna mimic/mirna inhibitor to transfect, refer to the manufacturer's instructions. Where possible, dilute mirna mimic/mirna inhibitor in the same volume as recommended for sirna in the protocol. Procedure 1. Spot ng sirna in 1 3 µl of sirna Suspension Buffer/RNase-free water into a single well of a 384-well plate (this will give a final sirna concentration of 5 nm after addition of cells to complexes in step 4). Note: After this step, sirna may be stored at 20 C for long time periods. sirna may be stored in solution or may be dried in the plate at room temperature (15 25 C) before storage. 2. Add 0.5 µl of HiPerFect Transfection Reagent to 9.5 µl of culture medium without serum. Add the diluted HiPerFect Transfection Reagent to the prespotted sirna. Note: To ensure accurate pipetting, diluted HiPerFect Reagent should be prepared in a larger volume for use in multiple wells (see Transfection in multiwell plates preparing a master mix on page 17). IMPORTANT: The amount of HiPerFect Transfection Reagent and sirna required for optimal performance may vary, depending on the cell line and gene target. For details on optimization of the sirna to HiPerFect Transfection Reagent ratio, see page Incubate for 5 10 min at room temperature (15 25 C) to allow formation of transfection complexes. 24 HiPerFect Transfection Reagent Handbook 10/2010

25 4. Seed ,000 cells in 40 µl of an appropriate culture medium (containing serum and antibiotics) into the well, on top of the sirna HiPerFect Reagent transfection complexes. 5. Incubate the cells with the transfection complexes under their normal growth conditions and monitor gene silencing after an appropriate time (e.g., 6 72 h after transfection, depending on experimental setup). Change the medium as required. Note: The optimal incubation time for gene silencing analysis depends on cell type, the gene targeted, and the method of analysis. This can be determined by performing a time-course experiment. 384-Well Plates HiPerFect Transfection Reagent Handbook 10/

26 Protocol: Transfection of Adherent Cells with sirna/mirna (Traditional Protocol) This protocol is provided as a starting point for optimization of sirna/mirna transfection of adherent cells in a single well of a 24-well plate using HiPerFect Transfection Reagent if seeding cells the day before transfection is preferred. Starting points for optimizing transfection in other formats are listed in Table 3 on page 16. For some cell types, such as HepG2, the Fast-Forward Protocol (page 20) may give higher gene silencing effects at very low sirna concentrations compared to this protocol. Traditional Protocol Important points before starting Cells should be in optimal physiological condition on the day of transfection. The optimal confluency for transfection of adherent cells using this protocol is 50 80%. The amount of cells seeded depends on the cell type and time of analysis. If sirna has been ordered from QIAGEN, it is delivered lyophilized and must be resuspended prior to transfection.to resuspend, follow the instructions provided with the sirna. Nucleic acid amounts in the protocol refer to sirna. For appropriate amounts of mirna mimic/mirna inhibitor to transfect, refer to the manufacturer's instructions. Where possible, dilute mirna mimic/mirna inhibitor in the same volume as recommended for sirna in the protocol. Procedure 1. The day before transfection, seed 2 8 x 10 4 cells per well of a 24-well plate in 0.5 ml of an appropriate culture medium containing serum and antibiotics. 2. Incubate the cells under normal growth conditions (typically 37 C and 5% CO 2 ). 3. On the day of transfection, dilute 37.5 ng sirna in 100 µl culture medium without serum (this will give a final sirna concentration of 5 nm after adding complexes to cells in step 5). Add 3 µl of HiPerFect Transfection Reagent to the diluted sirna and mix by vortexing. IMPORTANT: The amount of HiPerFect Transfection Reagent and sirna required for optimal performance may vary, depending on the cell line and gene target. For details on optimization of the sirna to HiPerFect Transfection Reagent ratio, see page Incubate the samples for 5 10 min at room temperature (15 25 C) to allow the formation of transfection complexes. 5. Add the complexes drop-wise onto the cells. Gently swirl the plate to ensure uniform distribution of the transfection complexes. 26 HiPerFect Transfection Reagent Handbook 10/2010

27 6. Incubate the cells with the transfection complexes under their normal growth conditions and monitor gene silencing after an appropriate time (e.g., 6 72 h after transfection, depending on experimental setup). Change the medium as required. Note: The optimal incubation time for gene silencing analysis depends on the cell type, the gene targeted, and the method of analysis. This can be determined by performing a time-course experiment. When using fluorescently labeled sirna, microscopic analysis should be performed 4 24 h after transfection. Traditional Protocol HiPerFect Transfection Reagent Handbook 10/

28 Protocol: Transfection of Suspension Cell Lines, Including Jurkat and K562, with sirna/mirna in 24-Well Plates This protocol is provided as a starting point for optimization of sirna/mirna transfection of suspension cells in a single well of a 24-well plate using HiPerFect Transfection Reagent. Details about optimization of transfection of suspension cells and starting points for transfection in different formats are provided in Appendix A (page 55). Suspension Cells Cultivation of suspension cells Optimal transfection efficiency is achieved when suspension cells are cultivated in a spinner flask for at least 24 hours prior to transfection. This ensures optimal gas exchange and that the cells are in logarithmic growth phase. The day before transfection, an aliquot of cells is mixed with trypan blue (which stains dead cells) and living (unstained) cells are counted using a counting chamber. An appropriate volume of cells is centrifuged and the cell pellet is resuspended in fresh culture medium and transferred to a spinner flask. The cell density in the spinner flask should be 3 x 10 5 cells per ml (protocol step 1). Spinner flasks are available in various sizes (e.g., 50 ml, 250 ml, and 1000 ml). Pendulums inside the flask stir the cell culture. The volume of medium in the flask should be approximately one third of the flask size (e.g., a 250 ml spinner flask should contain ~85 ml cell suspension). The cap of the flask should be loose to allow air to enter. The cell suspension should be constantly stirred at ~60 stirs per minute. The next day, cell number is determined. For most cell types, the cell number should have doubled. The appropriate volume of cells is then harvested for transfection (protocol step 3). If cells grow significantly slower than expected, it may indicate that cells are not healthy. In this case, repeat the overnight incubation with fresh cells and medium. Important points before starting Cells should be in optimal physiological condition on the day of transfection. For recommended cell numbers to seed, see Table 7, page 56. The optimal amount of cells to seed depends on the cell line and the time of analysis. If sirna has been ordered from QIAGEN, it is delivered lyophilized and must be resuspended prior to transfection.to resuspend, follow the instructions provided with the sirna. Nucleic acid amounts in the protocol refer to sirna. For appropriate amounts of mirna mimic/mirna inhibitor to transfect, refer to the manufacturer's instructions. Where possible, dilute mirna mimic/mirna inhibitor in the same volume as recommended for sirna in the protocol. 28 HiPerFect Transfection Reagent Handbook 10/2010

29 Procedure 1. The day before transfection, dilute cells at a density of 3 x 10 5 per ml in an appropriate culture medium containing serum and antibiotics in a spinner flask. 2. Incubate the cells under normal growth conditions (typically 37 C and 5% CO 2 ). 3. On the day of transfection, plate 2 x 10 5 cells per well of a 24-well plate in 100 µl culture medium containing serum and antibiotics. 4. Dilute 750 ng sirna in 100 µl culture medium without serum (this will give a final sirna concentration of 100 nm after adding medium in step 8). Add 6 µl HiPerFect Transfection Reagent to the diluted sirna and mix by vortexing. IMPORTANT: The amount of HiPerFect Transfection Reagent and sirna required for optimal performance may vary, depending on the cell line and gene target. 5. Incubate the samples for 5 10 min at room temperature (15 25 C) to allow the formation of transfection complexes. 6. Add the complexes drop-wise onto the cells. Gently swirl the plate to ensure uniform distribution of the transfection complexes. IMPORTANT: When performing transfections in 96-well plates, transfection efficiency can be significantly enhanced by thoroughly mixing the cells and transfection complexes by pipetting up and down 4 6 times. 7. Incubate the cells with the transfection complexes under their normal growth conditions for 6 h. 8. Add 400 µl culture medium containing serum and antibiotics to the cells and incubate until analysis of gene silencing (e.g., 6 72 h after transfection, depending on the experimental setup). Add fresh medium as required. Note: The optimal incubation time for gene silencing analysis depends on the cell type, the gene targeted, and the method of analysis. This can be determined by performing a time-course experiment. Suspension Cells HiPerFect Transfection Reagent Handbook 10/

30 Protocol: Transfection of Macrophage Cell Lines, Including J774.A1 and RAW 264.7, with sirna/mirna in 24-Well Plates This protocol is provided as a starting point for optimization of sirna/mirna transfection of macrophage cells in a single well of a 24-well plate using HiPerFect Transfection Reagent. Details about optimization of transfection of macrophage cells and starting points for transfection in different formats are provided in Appendix A (page 55). Important points before starting Cells should be in optimal physiological condition at the time of transfection. For recommended cell numbers to seed, see Table 7, page 56. The optimal amount of cells to seed depends on the cell line and the time of analysis. If sirna has been ordered from QIAGEN, it is delivered lyophilized and must be resuspended prior to transfection.to resuspend, follow the instructions provided with the sirna. Nucleic acid amounts in the protocol refer to sirna. For appropriate amounts of mirna mimic/mirna inhibitor to transfect, refer to the manufacturer's instructions. Where possible, dilute mirna mimic/mirna inhibitor in the same volume as recommended for sirna in the protocol. Macrophages Procedure 1. Shortly before transfection, seed x 10 5 cells per well of a 24-well plate in 100 µl of an appropriate culture medium containing serum and antibiotics. Cells may alternatively be seeded after step 3 of this protocol. 2. For the short time until transfection, incubate the cells under normal growth conditions (typically 37 C and 5% CO 2 ). 3. Dilute 375 ng sirna in 100 µl culture medium without serum (this will give a final sirna concentration of 50 nm after adding medium in step 7). Add 6 µl HiPerFect Transfection Reagent to the diluted sirna and mix by vortexing. IMPORTANT: The amount of HiPerFect Transfection Reagent and sirna required for optimal performance may vary, depending on the cell line and gene target. 4. Incubate the samples for 5 10 min at room temperature (15 25 C) to allow the formation of transfection complexes. 5. Add the complexes drop-wise onto the cells. Gently swirl the plate to ensure uniform distribution of the transfection complexes. 6. Incubate the cells with the transfection complexes under their normal growth conditions for 6 h. 30 HiPerFect Transfection Reagent Handbook 10/2010